Abstract

A simple, precise, rapid reproducible, selective, and stability indicating reverse phase UPLC method has been developed for the estimation of dronedarone in pharmaceutical dosage form. Estimation of dronedarone hydrochloride was achieved on Acquity BEH C18 () 1.7 μm column using buffer [20 mM KH2PO4 + 1 mL Triethylamine ( by orthophosphoric acid)] and methanol in ratio of 40 : 60 as mobile phase at 30°C. The flow rate was 0.4 mL/min and effluents were monitored at 290 nm. The method was validated with respect to linearity, accuracy, precision, LOD, LOQ, and robustness. The method was linear over the concentration range 0.38–90 μg/mL (), with a limit of detection and quantification of 0.1 and 0.38 μg/mL, respectively. Dronedarone was subjected to acid and alkali hydrolysis, chemical oxidation, dry heat degradation, and photo (sunlight) degradation. The degraded product peaks were well resolved from the drug peak with significant difference in their retention time value.

1. Introduction

Dronedarone (DRO), N (2Butyl3 (p (3(dibutylamino)propoxy)benzoyl)-5-benzofuranyl) methane sulfonamide (Figure 1). DRO is used as an alternative to amiodarone for the treatment of atrial fibrillation and atrial flutter in people whose hearts have either returned to normal rhythm or who undergo drug therapy or electric shock treatment to maintain normal rhythm [1]. A literature review revealed that DRO has been determined and studied by several procedures; quantitative analysis of DRO in bulk drug and pharmaceutical dosage is formed by HPLC [2–4] and by HPTLC [5]. The HPLC method for determination of DRO in plasma and myocardium has also been reported [6]. There is a report on the determination of the DRO in human plasma by liquid chromatography-tandem mass spectrometry [7]. There is no method reported for quantification of dronedarone by UPLC.

Figure 1

Structure of dronedarone hydrochloride.

2. Experimental

2.1. Material and Reagents

Pure DRO was procured as gift sample from Sanofi Aventis, India. DRO tablet was purchased from local commercial sources. HPLC grade methanol was procured from Merck India, Mumbai, India. Potassium dihydrogen phosphate and orthophosphoric acid (S.D. Fine Chemicals, Mumbai, India) were of analytical grade used for the preparation of mobile phase. HPLC grade water acquired from Millipore Milli-Q water system.

2.2. Chromatographic Conditions

Analysis of dronedarone was carried out on a Waters Acquity UPLC with PDA detector. The output signal was monitored and processed using empower software. The chromatographic column used was Acquity BEH C18 () mm and 1.7 μm particle size. Isocratic elution analysis was used throughout the analysis.

2.3. Mobile Phase

2.4. Instrument Parameter

The isocratic flow rate of mobile phase was maintained at 0.4 mL/min. The column temperature was adjusted to 35°C. The injection volume was set as 1 μL. Eluted sample was monitored at 290 nm and the run time was 3.0 min. The retention time of the DRO was about 1.5 min.

2.5. Preparation of Tablet Extract

10 tablets (each tablet contained 400 mg DRO) were weighed, transferred into a clean and dry mortar, and powdered. Tablet powder equivalent to 25 mg of DRO was transferred into a 50 mL volumetric flask and 30 mL of the methanol was added. The solution was sonicated for 15 min to achieve complete dissolution of DRO and then filtered through a Whatman filter paper number 42. Wash the residue with small amount of methanol and the volume was made up to the mark with the methanol.

Appropriate volume of the aliquot was transferred to a 50 mL volumetric flask, and the volume was made up to the mark with the mobile phase to obtain a solution containing 50 μg/mL of DRO.

3. Validation

3.1. Accuracy

To determine the accuracy, DRO solutions were analysed at three different concentrations (50%, 100%, and 150%). Mobile phase was used as a blank and injected before sample injection and the RSD values of peak area and retention time were calculated.

3.2. Precision

Precision study was performed by injecting the solution containing DRO (50 μg/mL) repeatedly, and peak area was measure. The results were reported in terms of relative standard deviation (Figure 2). The intraday and interday precision study of DRO was carried out by estimating the corresponding responses on the same day and on different days and the results were reported in terms of relative standard deviation (RSD).

Figure 2

Chromatogram of DRO reference standard.

3.3. Limit of Detection (LOD) and Quantification (LOQ)

The LOD and LOQ were determined at a signal-to-noise ratio of 3 and 10, respectively. LOQ and LOD were achieved by injecting a series of dilute solution of DRO. The precision and accuracy for DRO at LOQ were checked.

3.4. Linearity

Linearity was prepared from LOQ level to 160% of the actual sample concentration (50 μg/mL). A total of eight concentrations of the solutions were made separately and injected (LOQ: 40%, 60%, 80%, 100%, 120%, 140%, and 160% of the actual sample concentration). Calibration curve constructed by plotting peak area versus concentrations of the drug and regression equations was computed for DRO.

3.5. Robustness and Ruggedness

Robustness of the method was studied by deliberately changing the experimental conditions. The experimental conditions changed like flow rate ( mL/min), column oven temperature (°C), mobile phase composition (buffer: methanol = 38 : 62 and 42 : 58, v/v), and wavelength ( nm). Different columns of same dimensions were used for the analysis. The study was performed on the same day and on three different days by three different analysts. In each case, the RSD values were calculated for the peak area and retention time. The number of theoretical plates and tailing factors was compared with those obtained under the optimized conditions (Table 10).

3.6. Solution and Mobile Phase Stability

Stability of solution was investigated by injecting the sample into the chromatographic system in the time intervals of 0, 12, and 24 hrs. The mobile phase stability, was studied by injecting a freshly prepared sample solution at the same time intervals (0, 12, and 24 hrs) with the same mobile phase. The peak area was recorded and the RSD values were calculated.

3.7. Force Degradation Study

Stress degradation study using acid and alkali hydrolysis, chemical oxidation, dry heat degradation, and photo (sunlight) degradation was carried out and interference of the degradation products was investigate. All stress decomposition studies were performed at an initial drug concentration of 500 μg/mL in mobile phase and 5 mL of 1 M HCl, 1 M NaOH, and 5% H2O2 were added separately. The flasks were heated for 1 hr on a water bath at 80°C. The solutions were cooled and neutralized by adding acid or base, and then the solution was diluted up to the mark with diluent. Solid state thermal degradation was carried out by exposing drug to dry heat at 80°C for 48 hrs. For photolytic degradation studies, drug in solid state was exposed to sunlight for 48 hrs. The sample after stress degradation was used to prepare 50 μg/mL.

4. Result and Discussion

UPLC becomes very famous in the recent year due to its fast approach for method development and validation of pharmaceutical drug. The smaller particles in column increase the efficiency, resolution, and speed in analysis (Table 1). It is observed that the run time of the UPLC method is very short (3 min) to that of HPLC methods (Table 12). The present method discusses the development and validation of a method for quantitative analysis of DRO with degradation study under different stress conditions.

4.1. Method Optimization

Different chromatographic conditions were experimented to achieve better efficiency of the chromatographic system. Choice of retention time, tailing, theoretical plates, and run time was the major task while developing the method. Several proportions of buffer and solvents were evaluated in order to obtain suitable composition of mobile phase. In order to achieve symmetrical peak of DRO, various stationary phases like C8, C18 and phenyl column with different lengths (50 and 100 mm) were studied. From the summary, it is concluded that Acquity BEH C18 () mm, 1.7 μm column has the ideal stationary phase for the determination of DRO. UV spectra of DRO showed that the drug was absorbed appreciably at 290 nm, so the same was selected as the detection wavelength during the studies. Summary of mobile phase and stationary phase optimization is represented in Tables 2 and 3, respectively.

4.2. Accuracy

The accuracy of the method was determined by calculating recoveries of DRO. The recoveries for DRO obtained were 99.31%–99.98%. The results of accuracy study and RSD illustrate that the method is accurate. The summary of accuracy study is revealed in Table 4.

Level %

Number

Amount of drug added (g/mL)

Amount of drug found (g/mL)

Recovery (%)

Mean recovery (%)

RSD (%)

50

1

25.01

24.94

99.72

99.31

0.40

2

25.14

24.87

98.92

3

25.26

25.08

99.28

100

1

50.17

50.31

100.27

99.98

0.52

2

50.02

50.18

100.31

3

50.11

49.80

99.38

150

1

75.02

74.73

99.61

99.87

0.65

2

75.13

74.68

99.40

3

75.16

75.63

100.62

Table 4

Accuracy study of dronedarone hydrochloride.

4.3. Precision

The summary of precision study is illustrated in Table 5. In the intermediate precision, study results demonstrate that RSD values were in the same order of magnitude as those obtained for precision study. Overall, the data for the precision study suggested that assay value for each test preparation was between 98 and 102% and RSD of all results was less than 2%. Therefore, from all the observations, it can be concluded that the proposed method is highly precise.

Parameter

UPLC

Linearity (g/mL)

0.38–90

Correlation coefficient ()

0.999

Slope ()

2303

Intercept ()

2536

LOD (g/mL)

0.1

LOQ (g/mL)

0.38

Accuracy (% RSD)

0.40–0.65

Interday precision (RSD, )

0.59

Intraday precision (RSD, )

0.42

Table 5

Result of precision, linearity, LOD, and LOQ study.

4.4. Linearity

A calibration curve was obtained for DRO from LOQ to 180% of its actual sample concentration (50 μg/mL). A linear correlation was obtained between the mean peak area and the concentration in the range of 0.38–90 μg/mL, from which the linear regression equation was computed and found to be , (Figure 3). Where is the mean peak area, is the concentration of DRO in μg/mL and is the correlation coefficient. These results confirm the linearity relation between the mean peak area and concentration as well as the sensitivity of the method. The summary of linearity study is represented in Table 5.

Figure 3

Linearity study of DRO.

4.5. Robustness and Ruggedness

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate change in method parameters and provides an indication of its reliability during normal usage. On deliberate variation in chromatographic condition (flow rate, temperature, and mobile phase composition) of DRO analysis, the value of assay (with %RSD < 2), tailing factor, and theoretical plates remained near to those of the result obtained by the optimized chromatographic condition. The summaries of robustness and ruggedness studies are represented in Tables 6 and 7, respectively.

Parameter

Mean

% RSD

Mean area

% RSD

Flow rate

0.36

1.56

0.10

240218

0.27

0.40

1.52

0.14

232437

0.24

0.44

1.49

0.12

220445

0.28

Mobile phase composition (buffer : methanol)

(38 : 62)

1.41

0.14

217352

0.34

(40 : 60)

1.52

0.24

232416

0.27

(42 : 58)

1.58

0.11

245938

0.26

Column oven temperature

25°C

1.61

0.21

248475

0.39

30°C

1.52

0.28

232387

0.32

35°C

1.39

0.17

212868

0.21

Table 6

Result of robustness study.

Variable

% RSD

Area

% RSD

Theoretical plate

% RSD

Asymmetry

% RSD

Analyst

1.52

0.87

242374

1.48

3447

1.69

1.32

1.36

Column

1.52

0.42

242463

1.26

3559

1.82

1.28

1.22

Table 7

Ruggedness study of DRO.

4.6. Solution and Mobile Phase Stability

At the specified time interval, % assay of DRO obtained from drug solution stability and mobile phase stability was within accepted criteria. The results also confirmed that the standard solution of drug and mobile phase were stable at least for 24 hours during the assay analysis. The summary of solution and mobile phase stability is illustrated in Table 8.

Initial

6 hr

12 hr

24 hr

48 hr

% assay for solution stability

99.82

100.11

99.52

99.67

99.38

% assay for mobile phase stability

100.17

99.83

100.06

99.32

99.74

Table 8

Solution and mobile phase stability study.

4.7. Force Degradation Study

Force degradation study was carried out by subjecting the drug to acid, alkali, chemical oxidation, dry heat degradation, and photo (sunlight) degradation condition. The DRO was found to be stable to acid hydrolysis, dry heat degradation, and photolytic condition. Degradation was observed in alkali hydrolysis and oxidative stress condition. The summary of force degradation study is represented in Table 9. The figure of degradation study is represented in Figures 4, 5, 6, 7, 8, 9, and 10.

Stress condition

Time

Purity angle

Purity threshold

% degradation

Acid hydrolysis

5 hr

0.404

0.978

3.24

Base hydrolysis

1 hr

1.045

12.715

24.07

Oxidation

1 hr

3.297

11.91

18.56

Thermal

5 days

0.573

0.944

2.37

Photo

5 days

0.764

1.363

0.48

Table 9

Stress degradation study.

Parameter

UPLC

Retention time

1.52

Asymmetric factor

1.18

Theoretical plate

3482

Table 10

System suitability parameter.

Figure 4

Acid degradation study of DRO.

Figure 5

Alkali degradation study of DRO.

Figure 6

H2O2 degradation study of DRO.

Figure 7

Thermal degradation study of DRO.

Figure 8

Photo degradation study of DRO.

Figure 9

Peak purity result of alkali degradation.

Figure 10

Peak purity result of H2O2 degradation.

5. Conclusion

A stability-indicating reverse phase UPLC method has been developed and validated for the determination of DRO in bulk and their pharmaceutical dosage form. Compared to reported method [2–5], the proposed method is very short and sensitive. The result of stress testing undertaken according to the ICH guidelines revealed that the method is selective and stability indicating. Apart from alkali hydrolysis and oxidative stress degradation condition, the drug was found to be stable in the rest of forced degradation conditions. The proposed method was validated and found to be simple, accurate, precises and specific. The method was successfully applied for the estimation of tablet dosage form (Table 11). The proposed UPLC method can be applied for the analysis of sample obtained during stability study to predict expiration dates of pharmaceuticals. Hence, the method can be used in routine analysis of DRO drug in quality control laboratories.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

The authors are grateful to the Department of chemistry, SauraStra University, Rajkot, Gujarat, India for providing the laboratory facilities. They offer special thanks to the “National Facility for Drug Discovery through New Chemicals Entities (NCEs) Development and Instrumentation Support to Small Manufacturing Pharma Entities” Program under the Drug and Pharma Research Support (DPRS) jointly funded by Department of Science & Technology, New Delhi, Government of Gujarat, Industries Commissionerate and Saurashtra University, Rajkot, for providing instrument facility.

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